The association of collagen as a major structural material in a multiplicity of tissues, including bone, cartilage, skin, tendons, dentine and various soft tissues is well known. It is also known that the fiber structure of collagen is stabilized by crosslinking. The presence of the fluorescent pyridinium ring system as a non-reducible crosslink in collagen was reported by Fujimoto, D., et al., J Biochem (1978) 83:863-867. The Fujimoto paper reported isolation of a fluorescent peptide from pronase digestion of bovine Achilles tendon collagen. The isolated hydrolyzed pyridinoline (Pyd) was thought to contain three residues of hydroxylysine and it was recognized that, prior to hydrolysis, peptide fragments were attached to the pyridinoline moiety. Further work on characterization was conducted by Gunja-Smith, Z., et al., Biochem J (1981) 197:759-762, using hydrolyzed urine, and advantage was taken of the presence of the pyridinoline in urine by Robins, S. P., Biochem J (1982) 207:617-620, who linked pyridinoline obtained from hydrolyzed urine to a carrier to raise antibodies. The antibodies were then employed in an immunoassay to determine the concentration of pyridinoline in hydrolyzed urine. The procedure was stated by Robins as useful to provide an index of the degradation of certain forms of mature collagen by analysis of physiological fluids.
In all of the foregoing, hydrolyzates were employed to obtain total pyridinoline since much of the crosslink retained peptide extensions of the hydroxylysyl residues responsible for its formation. Thus, in order to obtain a homogenous preparation containing the pyridinium ring, a preliminary hydrolysis step was necessary.
By 1982, it was established that there were two pathways of crosslink formation depending on whether lysine or hydroxylysine residues were present in the telopeptides from which these crosslinks were derived (Robins, S. P., in "Collagen in Health and Disease" (1982) Weiss, J. B., et al., eds., pages 160-178, Churchill Livingstone, Edinburgh). This was stated to result in a specificity of crosslinking whereby in soft tissues, such as skin, reducible aldimine linkages are formed from oxidized lysyl residues, whereas in cartilage and bone these bonds, initially formed from hydroxylysine aldehydes, undergo a spontaneous rearrangement to more stable oxoimine crosslinks. These bonds undergo further reaction to form 3-hydroxy-pyridinium crosslinks. The stable crosslinking pyridinoline analog involving lysine rather than hydroxylysine in the helix portion was identified and quantified by Ogawa, T., et al., Biochem Biophys Res Commune (1982) 107:1251-1257; Eyre, D. R., et al., Anal Biochem (1984) 137:380-388, and designated deoxypyridinoline (Dpd). This material was then believed to be restricted to bone collagen, although amounts vary between species.
Further work by Robins, S. P., Biochem J (1983) 215:167-173, provided evidence for the existence of glycosylated pyridinoline in bone. Robins proposed a structure which showed the derivation of the ring from three residues of hydroxylysine and also showed that alkali hydrolyzates of collagen provided an O-galactosyl derivative substituted at the sidechain hydroxy group. As this material was extremely labile to mild acid treatment, this material would not have been present in samples of hydrolyzed tissue or body fluid.
Fujimoto, D., et al., J Biochem (1983) 94:1133-1136, chromatographed unhydrolyzed urine samples and showed that the 3-hydroxypyridinium ring portion was present in substantial proportion as the "free" form, i.e., the three hydroxylysyl-derived residues which composed it did not contain further peptide extensions. On amino acid analysis, whereas pyridinoline isolated from an acid hydrolyzate of collagen gave an asymmetric peak, "free" urinary pyridinoline gave a symmetric peak. The authors concluded this to be due to isomerization by epimerization of the hydroxylysine moiety of the pyridinoline system during hydrolysis. In addition, relationship of levels of total pyridinoline (after hydrolysis) to age was determined by these workers as a ratio to creatinine levels. It was found that the ratio was high in the urine of children but decreased with age until growth ceases. It was further found that this ratio is relatively constant in adults, but increases slightly in old age. The authors speculate that this may correspond to the loss of bone mass observed in old age.
Attempts were also made to characterize the above-mentioned peptide extensions. Robins, S. P., et al., Biochem J (1983) 215:175-182, proposed that in cartilage-derived type II collagen, the pyridinoline links two C-terminal telopeptide chains with a single chain of the helical peptide. An additional pyridinoline crosslink, i.e., with the ring derivatized to other peptides, was thought to link two N-terminal non-helical peptides with a third chain in the helical portion of the molecule. The studies were conducted by isolating the fluorescent pyridinoline crosslinks from tissues by specific cleavage with CNBr, thus preserving peptide sequences as extensions of the hydroxylysyl residues forming the ring. The crosslink was localized in the collagen fibers by determining the amino acid sequences of these extensions.
In a paper similar in approach to that of Robins (supra), Wu, J. J., et al., Biochemistry (1984) 23:1850-1857, conducted CNBr cleavage of mature cartilage and determined the sequence of the peptide extension residues of the hydroxylysyl participants in the pyridinium ring. Their conclusions were similar to those of Robins.
Robins, S. P., et al., Biochim Biophys Acta (1987) 914:233-239, used CNBr digestion of bone derived collagen to localize the crosslinks in the type I collagen structure. These authors concluded that the proportions of the crosslink derived from lysine and that derived from hydroxylysine were present in the same proportions in each of the isolated peptide forms. They also concluded that this showed that these two crosslink analogs occupy the same loci in the collagen fiber and that the form apparently derived from one lysyl participant appears to arise through incomplete hydroxylation of the appropriate lysine residues in the helix. Amino acid analysis indicated that the crosslinks must be situated at two locations involving both the N- and C- terminal telopeptide regions.
Henkel, W., et al., Eur J Biochem (1987) 165:427-436, determined the amino acid sequences associated with the crosslinks in type I collagen isolated from aorta. These sequences are different from those obtained for type II collagen. Similar results were found by Eyre, D. R., et al., FEBS (1987) 2:337-341, who demonstrated that the crosslinks from type IX and type II collagens displayed distinctive peptides attached to the pyridinoline crosslinks.
PCT application WO89/04491 to Washington Research Foundation proposes a urinary assay for measuring bone resorption by detection in urine of the specific crosslinks, characterized by their peptide extensions, associated with bone collagen. The assay relies on quantifying the concentration of peptides in a body fluid where the peptide fragments having a pyridinium crosslink are derived from bone collagen resorption. Two specific entities having peptide extensions presumed to be associated with bone collagen are described. These are obtained from the urine of patients suffering from Paget's disease, a disease known to involve high rates of bone formation and destruction.
Macek, J., et al., Z Rheumatol (1987) 46:237-240, proposed an assay for osteoarthrosis which depends upon the peptides associated with the crosslinks from collagen breakdown. In this approach, the urine sample was size-separated for peptides of molecular weight greater than 10 kd, which peptides were then separated by HPLC using a fluorescence detector to determine those fractions containing the fluorescence due to the pyridinium ring. The spectra obtained from patients with osteoarthrosis were compared to those from healthy patients, and it was easily demonstrable that the multitude of fluorescent peaks associated with the diseased condition was absent from the healthy counterpart. Furthermore, urine from the same diseased patient two weeks after total endoprosthesis of the diseased hip, thereby decreasing the products of osteoarthrosis, gave a spectrum of fluorescent peaks which more closely resembled that of normals. Furthermore, the osteoarthrosis spectrum was readily distinguished from that obtained from patients with rheumatoid arthritis. The closer resemblance of the rheumatoid arthritis spectrum to that of the spectrum from normal controls was attributed by the authors to the higher activity of proteases in rheumatoid arthritis. This was presumed to digest collagen structures into smaller fragments not detectable in their system.
Study of the elevated levels of total 3-hydroxypyridinium ring crosslinks in hydrolyzed urine of patients with rheumatoid arthritis has also been suggested as a method to diagnose this disease by Black, D., et al., Annals of Rheumatic Diseases (1989) 48:641-644. The levels of "hydrolyzed" crosslink for patients with rheumatoid arthritis (expressed as a ratio of this compound to creatinine) were elevated by a factor of 5 as compared to controls. In this method, crosslinks derived from hydroxylysine were distinguishable from those derived from lysine; only the hydroxylysine-derived crosslinks were measurably increased. In a more extensive study using hydrolyzed urines, Seibel et al., J Rheumatol (1989 16:964-970, showed significant increases in the excretion of bone-specific crosslinks relative to controls in both rheumatoid and osteoarthritis, but the most marked increases for hydroxylysine-derived pyridinium were in patients with rheumatoid arthritis.
While measures related to the presence of collagen-derived crosslinks have been used as indices of the degradation of specific collagen types, including that of bone, conversely, efforts have been made to identify markers of bone formation. Delmas, P. D., et al., J Bone Mineral Res (1986) 1:333-337, used the level of GLA- protein in serum as a marker for bone formation in children; the same group, Brown, J. P., et al., used a similar assay to assess bone formation in post-menopausal osteoporosis (Lancet (1984) 1091-1093.
There are many conditions in humans and animals which are characterized by a high level of bone resorption and by an abnormal balance between bone formation and bone resorption. Among the best known of these are osteoporosis and Paget's disease. However, abnormalities in bone metabolism occur in a variety of other conditions including the progress of benign and malignant tumors of the bone and metastatic cancers which have been transferred to bone cells from, for example, prostate or breast initial tumors. Other conditions include osteopetrosis, osteomalacial diseases, rickets, abnormal growth in children, renal osteodystrophy, and a drug-induced osteopenia. Irregularities in bone metabolism are also often side effects of thyroid treatments and thyroid conditions per se, such as primary hypothyroidism and thyrotoxicosis as well as Cushing's disease. It would be useful to have a diagnostic which readily recognizes a subject's condition as an irregularity in bone metabolism, even without defining the precise syndrome from among the possible choices, such as those listed here. Additional tests within the sphere of known bone diseases can be performed once it is established that this is the subset of problems from which diagnosis will emerge.
The invention provides just such a screening test, which is general for bone metabolism abnormalities.